1. Field of the Invention
This invention relates to the field of antisense oligonucleotides for use in the inhibition of vascular endothelial cell growth factor (VEGF) expression.
2. Description of Related Art
Vascular endothelial cell growth factor (VEGF), also known as vascular permeability factor (VPF), is a 34-43 kDa (with the predominant species at about 45 kDa) dimeric, disulphide-linked glycoprotein synthesized and secreted by a variety of tumor and normal cells. Leung et al., Science 246, 1306 (1989), observed three VEGF transcripts (121, 165, and 189 amino acids long, respectively), suggesting that an alternative splicing mechanism is involved. More recently, Houck et al. discovered a fourth VEGF transcript having a length of 206 amino acids. Tischer et al., J. Biol. Chem. 266, 11947 (1991), have determined that the human VEGF coding region is comprised of eight exons. Furthermore, this group proved that three mRNA transcripts (encoding for the 121, 165, and 189 amino acid long peptides) were the result of alternative splicing. Transcripts analogous to the 121 and 165 amino acid polypeptides have been identified in the bovine system. Leung et al., supra. The transcript corresponding to the 165 amino acid transcript have also been identified in the rodent systemxe2x80x94rat (Conn et al., Proc. Natl. Acad. Sci. U.S.A. 87, 2628 (1989)), guinea pig (Sanger et al., Cancer Res. 50, 1774 (1990)), and mouse (Claffey et al., J. Biol. Chem. 257, 16317 (1992)).
Tischer et al., supra, reported the nucleic acid sequence for three forms of human VEGF coding region. Claffey et al., supra, published the sequence for murine VEGF. Comparisons have revealed greater than 85% interspecies conservation of the VEGF molecule. All the alternatively spliced VEGF molecules have not been identified yet, but based on the conservation between species, they should be in the near future.
The following discussion presents several pathological states in which VEGF is involved and emphasizes the importance of VEGF as a potential target for therapeutic treatment.
Diabetic Retinopathy
Diabetic retinopathy is the leading cause of blindness among working age adults (20-64) in the United States. During the course of Diabetes Mellitus, one complication that can arise is an occlusion of the retinal veins. This venous occlusion results in the formation of microaneurysms due to the expansion of the vessel wall, hemorrhaging (leaking of blood into surrounding areas), xe2x80x9ccotton woolxe2x80x9d spots representing cellular exudates (i.e., cellular damage) and neovascularization of the retina extending into the vitreous, resulting in bleeding. Classic treatments for diabetic retinopathy are 1) the control of blood glucose and blood pressure and 2) pan retinal laser photocoagulation (PRP). Treatment #1 can prolong the onset of the disease depending on the diligence of the affected individual. Treatment #2 is quite effective, but can lead to additional hemorrhaging as well as damage to critical areas needed for visions (i.e., foveal fibers). Additional treatments for this disease which have less side effects would prove extremely valuable.
Recent observations have shown an increase in VEGF protein levels in retinal membranes from patients with diabetes, suggesting that this cytokine/growth factor may play an important role in the disease. The following characteristics of VEGF provide evidence that it may be an important regulator of diabetic retinopathy: (1) The action of VEGF is specific for endothelial cells; (2) VEGF has been shown to be angiogenic as well as mitogenic; (3) VEGF is a secreted molecule; (4) VEGF induces vascular permeability; and 5) VEGF is induced under hypoxic conditions (i.e., during retinal vein occlusion).
Atherosclerotic Plaque Formation
VEGF may play a role in the development of an atherosclerotic plaque. Atherosclerosis describes a state where the formation of lipid-containing lesions occurs in medium and large arteries. It is the primary cause of myocardial and cerebral infarctions in the United States. Lesions form within the intima, the innermost layer of the arterial wall, and are separated into two forms: the fatty streak (early), and the fibrous plaque (advanced). Both of these forms are characterized by lipid-filled macrophages (derived from blood-borne monocytes) and smooth muscle cells. The fibrous plaque is further characterized by the deposition of connective tissue and cholesterol crystals. These lesions occlude the lumen of the blood vessel diminishing the blood flow, leading to ischemia and necrosis. Research has shown that neovascularization can also occur in the atherosclerotic lesion. Levels of VEGF protein in these affected areas have not been determined, but it has been shown that both monocytes and macrophages express VEGF.
Wound Healing
VEGF may also be important in maintaining normal states of wound healing. See Brown et al., J. Exp. Med. 176, 1375 (1992). Wound healing is usually a regulated response to injury or trauma. Focal hemorrhaging is followed by the extravasation (leaking) of fibrinogen from the plasma to form a fibrin gel or clot. This initial matrix is replaced by granulation tissue (fibronectin, collagen, proteoglycan) and finally by scar tissue. In addition, keratinocytes migrate and form a covering to protect against fluid loss and bacterial infection. One major characteristic of wound healing is that vessel hyperpermeability occurs for some time after bleeding has stopped. In addition, angiogenic activity is detectable during this time period. Recent work has shown that keratinocytes, located at the border of the wound as well as in the wound covering, produce VEGF. Brown et al., supra. This result suggests that VEGF may be responsible for hyperpermeable and angiogenic activity associated with wound healing.
Aberrant would healing associated with surgery can result in complications such as hypertrophic scarring (excessive collagen deposition), keloid formation (scar tissue invading normal surrounding tissue), and adhesions in the peritoneal cavity. Other problems related with unregulated wound healing occur during the formation of lung fibrosis and in diabetes mellitus (wounds do not heal). It is believed that VEGF plays a role in these processes as well.
Tumor Angiogenesis
VEGF may be a tumor angiogenesis factor. Plate et al., Nature 359, 845 (1992). Angiogenesis is the tightly regulated processes by which new blood vessels develop. The development of a vascular system is necessary for the flow of nutrients and waste to and from tissues and organs. Smaller solid tumors ( less than 1-2 mm) do not require an extensive vascular system to survive, but instead derive their nourishment through the diffusion of needed nutrients. However, in order for these cell masses to grow beyond several millimeters in size, additional vascularization is needed. See, e.g., Folkman, J. Natl. Cancer Inst. 82, 4 (1990). It has been suggested that inhibition of tumor angiogenesis might be an effective strategy to combat tumor growth and circumvent acquired resistance to traditional anti-cancer therapeutic agents. Kerbel, BioEssays 13, 31(1991). Kim et al., Nature 362, 841 (1993) reported that monoclonal antibodies specific for VEGF inhibited the growth of tumors in vivo.
The tumor stroma, which contains both connective tissue and the vascular system, is essentially the xe2x80x9clifelinexe2x80x9d of the tumor. Whereas normal tissue vasculature is organized and can respond to changes in metabolism, the tumor stroma is poorly organized and closely resembles scar tissue found during wound healing. The tumor stroma may represent only a small portion of the total tumor (e.g., medullary carcinoma of the breast) or may exist as 80-90% of the total cell mass (e.g., desmoplastic carcinoma). Tumor blood vessels also differ from those found in normal tissue in that they are hyperpermeable to plasma and plasma proteins. Whereas this porosity is seen in normal tissue only during wound healing, solid tumors maintain this porous characteristic indefinitely.
While a necessary component for tumor growth, the stroma also acts as a barrier against macromolecules (e.g., monoclonal antibodies) which are needed in sufficient quantities to be effective as therapeutic agents. In large tumors, antibodies/macromolecules may not be effective due to large diffusional spaces as well as absorption into perivascular regions of peripheral tumor cells. Consequently, an alternative therapeutic compound is desirable.
As just discussed, VEGF is principal component in many pathological states and processes. Research has shown that VEGF is present in regions of tumors where capillary growth is occurring and suggests that VEGF can trigger the entire sequence of events leading to angiogenesis. By contrast, VEGF levels in normal tissues is relatively low. Regulation of the levels of VEGF expression, therefore, could prove to be an important method of treating pathological conditions without significantly affecting normal tissue. For instance, it follows from the earlier discussion that inhibition of VEGF expression may play an important role in (a) regulating the ocular complications associated with diabetic retinopathy, (b) regulating the formation of an atherosclerotic plaque, (c) controlling certain unregulated instances relating to wound healing processes, and (d) preventing and altering angiogenesis associated with tumor growth and metastasis. These, of course, are but examples of the diseased states in which VEGF is involved and for which regulation of VEGF expression could prove useful. Other pathologic states brought about (in part) by VEGF expression are also potential candidates for treatment by regulation of VEGF expression.
Antisense oligonucleotide technology may provide a novel approach to the inhibition of VEGF expression. See generally Agrawal, Trends in Biotech. 10, 152 (1992). By binding to the complementary nucleic acid sequence (the sense strand), antisense oligonucleotides are able to inhibit splicing and translation of RNA. In this way, antisense oligonucleotides are able to inhibit protein expression. Antisense oligonucleotides have also been shown to bind to genomic DNA, forming a triplex, and inhibit transcription. Furthermore, a 17-mer base sequence statistically occurs only once in the human genome, and thus extremely precise targeting of specific sequences is possible with such antisense oligonucleotides.
In 1978 Zamecnik and Stephenson were the first to propose the use of synthetic antisense oligonucleotides for therapeutic purposes. Stephenson and Zamecnik, Proc. Natl. Acad. Sci. U.S.A. 75, 285 (1978); Zamecnik and Stephenson, Proc. Natl. Acad. Sci. U.S.A. 75, 280 (1978). They reported that the use of a oligonucleotide 13-mer complementary to the RNA of Rous sarcoma virus inhibited the growth of the virus in cell culture. Since then, numerous other studies have been published manifesting the in vitro efficacy of antisense oligonucleotide inhibition of viral growth, e.g., vesicular stomatitis viruses (Leonetti et al., Gene 72, 323 (1988)), herpes simplex viruses (Smith et al, Proc. Natl. Acad. Sci. U.S.A. 83, 2787 (1986)), and influenza virus (Zerial et al., Nucleic Acids Res. 15, 9909 (1987)).
Antisense oligonucleotides have also been shown to inhibit protein expression in mammalian systems. For example, Burch and Mahan, J. Clin. Invest. 88, 1190 (1991), disclosed antisense oligonucleotides targeted to murine and human IL-1 receptors that inhibited IL-1-stimulated PGE2 synthesis in murine and human fibroblasts, respectively; Colige et al., Biochemistry 32, 7 (1993) disclosed antisense oligonucleotides that specifically inhibited expression of a mutated human procollagen gene in transfected mouse 3T3 cells without inhibiting expression of an endogenous gene for the same protein; and Monia et al., J. Biol. Chem. 267, 19954 (1992), disclosed selective inhibition of mutant Ha-ras mRNA expression with phosphorothioate antisense oligonucleotide.
In most cases, however, unmodified antisense oligonucleotides are unsuitable for use in in vivo systems because of their susceptibility to attack by nucleases. Consequently, there has been much research in the area of modifying oligonucleotides to make them immune to such attack, thereby stabilizing the molecules for in vivo use. See generally Uhlmann and Peymann, Chemical Reviews 90, 543 (1990) at pages 545-561 and references cited therein. Focus has been on modifying the internucleotide phosphate residues, modifying the nucleoside units, modifying the 2xe2x80x2 position and substituting other moieties for the internucleotide phosphate. For example, Padmapriya and Agrawal, Bioorg. and Med. Chem. Lett. 3, 761 (1993) disclosed synthesis of oligodeoxynucleoside methlyphosphonothioates; Temsamani et al., Ann. N.Y. Acad. Sci. 660, 318 (1992) disclosed certain 3xe2x80x2 end-capped oligodeoxynucleotide phosphorothioates; and Tang et al., Nucleic Acids Res. 21, 2729 (1993) disclosed self-stabilized antisense oligodeoxynucleotide phosphorothioates having a hair-pin loop structure at their 3xe2x80x2 ends.
Many modified antisense oligonucleotides are capable of withstanding nucleolytic degradation, yet are still capable of hybridizing to target sequences and, thus, inhibiting protein expression. These modified oligonucleotides are better suited for in vivo applications. Tang et al., supra, showed that self-stabilized antisense oligonucleotides showed greater in vivo stability than their linear counterparts in mice. Simons et al. Nature 359, 67 (1992) reported the use of two antisense c-myb phosphorothioate oligonucleotides that suppressed intimal accumulation of rat carotid arterial smooth muscle cells in vivo.
The oligonucleotides disclosed by Pederson et al. in U.S. Pat. No. 5,220,007 (""007) is another modified antisense oligonucleotide that may be particularly well-suited for both in vitro and in vivo inhibition of protein expression. That molecule comprises an internal sequence having two or more consecutive, modified or unmodified, phosphodiester linkages. The internal sequence is flanked on both sides by modified nucleic acid sequences. The internal sequence activates RNase H, while the flanking sequences are unable to activate RNase H. The result is that when the oligonucleotide of the ""007 patent is bound to the target mRNA sequence, RNase H will excise the region of the target sequence complementary to the internal sequence of the antisense oligonucleotide. The target mRNA is thereby inactivated and protein expression inhibited.
Similarly, 3xe2x80x2 end-capped (Temsamani et al., supra) and self-stabilized 3xe2x80x2 hair-pin loop (Tang et al., supra) antisense oligonucleotides have been shown to have increased stability to nucleolytic attack and therefore may be well suited for inhibition of protein expression. The 3xe2x80x2 hair-pin loop structure of Tang et al. is characterized as having a 3xe2x80x2-terminal sequence that is substantially complimentary and anneals to an internal sequence.
There is another convincing rational behind the use of antisense oligonucleotide inhibition of VEGF expression to control angiogenesis. Whereas macromolecules such as monoclonal antibodies may have difficulty in reaching their target site at an effective concentration, antisense oligonucleotides can more easily enter cells/cell masses and accumulate at inhibiting concentrations. Antisense inhibition of VEGF is likely to provide an important tool in altering the development of abnormal angiogenesis.
Inhibition of VEGF expression by means of antisense oligonucleotide technology will also be useful in determining the role of this cytokine in processes where angiogenesis is involved. In vitro systems which mimic blood vessel formation/permeability have been developed. The role of VEGF in these systems can be determined using antisense oligonucleotides. Other in vitro systems, in use or being designed, can benefit from this technology. There are several areas where the role of VEGF has not been determined. If inhibition of VEGF does not reduce tumor growth, it does not mean other systems (psoriasis, fertilizations-implantation, vascularization of the endometrium) should not be investigated.
Vascular Endothelial Growth Factor (VEGF) has been shown to play an integral role in angiogenesis associated with a variety of pathological conditions. An object of the present invention is to suppress angiogenesis associated with pathological conditions. A further object of the present invention is to provide useful compounds, compositions and methods for preventing the expression of VEGF associated with these states. A still further object of the present invention is to provide compounds, compositions and methods for the treatment of these pathological states.
Accordingly, this disclosure presents antisense oligonucleotides that have been constructed and are targeted to bind to nucleic acid sequences encoding VEGF, thereby blocking production of the expression product. Also presented are methods for inhibiting VEGF expression and angiogenesis using these oligonucleotides, both in vitro and in vivo.